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PubMed2002

Characterization of a novel cellular retinoic acid/retinol binding protein from shrimp: expression of the recombinant protein for immunohistochemical detection and binding assay.

Members of the cellular retinoic acid (CRABP) and retinol binding (CRBP) proteins family are involved in the metabolic pathways of retinoic acid (RA) and retinal respectively. The objective of this study is to determine whether such proteins are present in crustaceans. We report here the cloning and isolation of a novel complementary DNA (cDNA) that showed characteristics of the CRABP/CRBP from the ovary and eyestalk of the shrimp. The cDNA is 0.9 Kb in size and the deduced shrimp protein is encoded for a protein of 14 kDa. Although it shows high amino acids sequence similarity to both the vertebrate and invertebrate CRABP, some conserved amino acids identified in other CRABPs were not found in MeCRABP. MeCRABP is expressed in the ovary, eyestalk, testis, epidermis and early larvae. The presence of MeCRABP in early larval stages suggests that the protein may be involved in the early larval development. Recombinant MeCRABP was produced and used to generate a polyclonal antibody. In the immunohistochemical detection study, anti-rCRABP antibody recognized the presence of CRABP in several cell types of the eyestalk as well as the smaller oocytes of the ovary. Although MeCRABP messenger RNA transcripts can be detected in the ovary throughout the ovarian maturation period, CRABP was detected only in the primary oocytes of the ovary. The results suggest that CRABP transcripts in the mature ovary are not translated and may be supplied to the oocyte as maternal messages. The binding property of the recombinant MeCRABP was also tested by a fluorometeric method. The result indicates that rMeCRABP binds to both RA and retinal with similar affinity. This study represents the first cloning and characterization of a cDNA that belongs to a member of retinoid/fatty acid binding protein family in crustaceans.

Amino Acid Sequence↗

PubMed2002

Molecular cloning, mapping and characterization of a novel mouse RING finger gene, Mrf1.

With a combined approach of database search, heterologous polymerase chain reaction (PCR), reverse transcription-PCR, rapid amplification of complementary DNA ends and genomic library screening, we have successfully cloned a mouse RING finger gene, mouse RING finger 1 (Mrf1). The Mrf1 gene has two exons of 63 and 2665 bp, respectively, and one intron of over 13 kb. An open reading frame was identified exclusively in exon 2, which encodes a putative protein of the RING-B box-coiled coil or the tripartite motif type of 403 amino acids. Mrf1 is moderately expressed in the spleen, brain and heart as a single 3.0 kb product and very highly expressed in the testis as two transcripts of 3.0 and 1.5 kb, respectively. The Mrf1 gene was mapped to mouse chromosome 3, between markers D3Mit70 and D3Mit277. Western blotting analysis indicated that an expected protein of approximately 44 kD was detected in the brain extracts of mouse, rat and human. The possible functions of Mrf1 are discussed in the contexts of protein-protein interactions, oncogenesis and ubiquitination.

Amino Acid Sequence↗

PubMed2002

The human gamma-aminobutyric acid A receptor delta (GABRD) gene: molecular characterisation and tissue-specific expression.

Terminal deletions of 1p36 result in a specific and common syndrome characterised by the following: growth delay, distinctive facial anomalies, hearing and visual deficits, heart defects, body asymmetry, moderate to severe psychomotor retardation, epilepsy, and self-abusive behaviour. The human gamma-aminobutyric acid A receptor delta-subunit gene (GABRD) encodes for one of at least 15 ligand-gated chloride channels for gamma-aminobutyric acid (GABA), the major inhibitory neurotransmitter in the mammalian brain. Recently we have mapped this gene by radiation hybrid mapping to the critical region of gene loss of the 1p36 deletion syndrome within 1p36.33. The complete complementary DNA (cDNA) sequence of GABRD was generated using assembled sequence of cDNA fragments already available, and 5'-rapid amplification of cDNA ends products. Fine physical mapping of the GABRD gene within this genomic interval was performed by screening bacterial artificial chromosome contigs spanning the critical region of the 1p36 deletion syndrome. The GABRD gene maps immediately proximal to the PRKCZ gene that is located between marker D1S243 and cosmid D1Z2--a region thought to be critical for cognition and speech development. The GABRD gene is expressed most abundantly in brain and has three alternative exons (1A-C) with alternative start codons at the 5'-end. Genomic localisation, function, and expression would suggest that the GABRD gene represents a good candidate for the neurodevelopmental and neuropsychiatric anomalies seen in the 1p36 deletion syndrome.

Amino Acid Sequence↗

PubMed1999

The mammalian gene collection.

The Mammalian Gene Collection (MGC) project is a new effort by the NIH to generate full-length complementary DNA (cDNA) resources. This project will provide publicly accessible resources to the full research community. The MGC project entails the production of libraries, sequencing, and database and repository development, as well as the support of library construction, sequencing, and analytic technologies dedicated to the goal of obtaining a full set of human and other mammalian full-length (open reading frame) sequences and clones of expressed genes.

Animals↗

PubMed2000

Polymorphisms of RhD(Va) and a new RhD(Va)-like variant found in Japanese individuals.

BACKGROUND AND OBJECTIVES: Red cell type RhD(Va) lacks epD1 and 5 and is encoded by hybrid RHD-CE(5)-D alleles. We analyzed RhD(Va) and RhD(Va)-like samples in Japanese blood donors. MATERIALS AND METHODS: Ten RhD(Va) samples lacked epD1 and 5 and 3 RhD(Va)-like variants also lacked, epD2 and a part of 6/7. We identified the full-length nucleotide sequences of the complementary DNA (cDNA) synthesized from 4 samples: 3 of type D(Va) and the 4th a D(Va)-like variant. RESULTS: Although their sequences differed from each other, all the substitutions were exclusively in exon 5. Three D(Va) samples had hybrid RHD-CE(5)-D alleles, but the D(Va)-like variant had a unique nucleotide substitution with a single amino acid change, E233K. Exon 5 of the genomic DNA from all 13 samples was analyzed by sequencing. No other sequences were identified. CONCLUSION: All RhD(Va) and RhD(Va)-like variants had the substitution for E233. E233 seems to be a determinant of epD1 and 5. A new category of RhD variant, DYO, was identified.

Amino Acid Substitution↗

PubMedDate not supplied

Structure of amylase genes in populations of Pacific Cupped oyster ( Crassostrea gigas): tissue expression and allelic polymorphism.

Using the previously determined complementary DNA Sequence of Crassostrea gigas amylase (Y08370), we designed several oligonucleotide primers and used them with polymerase chain reaction (PCR) technology to characterize oyster amylase gene sequences. Two genes encoding 2 different amylases were characterized and sequenced. The 2 genes are similarly organized with 8 exons and 7 introns. Intron insertions are found at the same location in the 2 genes. Sizes and nucleotide sequences are different for the different introns inside each gene and different for the corresponding introns in the 2 genes. Comparing the 2 genes, around 10% of the nucleotides are different along the exons, and comparing the 2 deduced protein sequences, a mean value of 10.4% of amino acids are changed. Genes A and B encode mature proteins of, respectively, 500 and 499 amino acids, which present 94% similarity. A microsatellite (TC(37)) that constitutes the largest part of intron 4 of gene A has been used as a polymorphic marker. A method consisting of a PCR step followed by EcoRI digestion of the obtained fragments was used to observe polymorphism in these 2 genes. Six and 4 alleles for genes A and B, respectively, have been sequenced, leading to a maximum of 2.9% base change. The 2 genes are ubiquitously expressed in the different digestive tissues with quantitative differences. Gene A is strongly expressed in the digestive gland and at a lower level in stomach, while gene B is preferentially expressed in the labial palps. The microsatellite repeat was used in the analysis of 4 populations of Crassostrea gigas from the French Atlantic coast. A high level of polymorphism observed with 30 different alleles of gene A inside the populations should allow their characterization using the mean value of the microsatellite allelic distribution. These populations showed a low level of differentiation ( F(st) between 0 and 0.011); however, the population of Bonne Anse appeared to be distinguished from the other populations.

Alleles↗

PubMed2000

Coding sequence, genomic organization and expression of a folate binding protein gene in the rat.

The complementary DNA (cDNA) and the gene encoding the folate binding protein alpha isoform (FBPalpha) have been reported for the human and mouse protein. However, there is no information about this gene in the rat, an animal that could be a model to study expression of this protein in vivo when folate metabolism is modified. Accordingly, the cloning and characterization of this gene in the rat have been the subject of this research. The gene has seven exons and six introns and is approximately 10kb in size. The organization and nucleotide sequence of the coding exons are similar to those of the corresponding human and mouse genes, which are the only other mammalian FBP genes cloned. However, the amino acid sequence of the rat FBPalpha is less homologous, having 48% identity with the published sequences for all the mammalian FBP isoforms. A finding not previously reported is the expression of two FBPalpha transcripts in the kidney that differ in the length of the 5' untranslated sequences, as determined by rapid amplification of cDNA end-polymerase chain reaction amplification (RACE-PCR). The brain expresses a single transcript intermediate in size between the two transcripts expressed in the kidney. The kidney transcripts are encoded by the same gene and appear to be regulated either from two independent promoters or from a single promoter in association with alternative RNA splicing.

Amino Acid Sequence↗

PubMed2002

Characterization and expression analyses of the mouse Wiskott-Aldrich syndrome protein (WASP) family member Wave1/Scar.

Characterization of multiprotein complexes involved in actin remodeling and cytoskeleton reorganization is essential to understand the basic mechanisms of cell motility and migration. To identify proteins implicated in these processes, we have isolated the mouse Wave1/Scar gene, a member of the Wiskott-Aldrich syndrome protein (WASP) family. The mouse Wave1 gene was physically localized on chromosome 10 and spans over 12 Kb comprising eight exons and seven introns. The mouse Wave1 complementary DNA encodes a predicted 559 amino acid protein, with a SCAR homology domain, a basic domain, a proline-rich region, a WASP homology domain and an acidic domain conserved in the orthologous proteins. The Wave1 transcription initiation site was mapped 210 base pairs upstream of the ATG translational start site. The presumptive proximal promoter contains putative consensus binding sites for E2 basic helix-loop-helix transcription factors, hepatocyte nuclear factor-3beta, S8 homeodomain protein, zinc finger transcription factor MZF-1, and an interferon-stimulated response element. Northern analysis demonstrated a strong expression of a unique approximately 2.6 Kb Wave1 transcript in brain tissue, and in situ hybridization showed restricted expression to Purkinje cells from the cerebellum and pyramidal cells from the hippocampus. Characterization and expression analyses of the murine Wave1 gene provide the basis toward functional studies in mouse models of the role of Wave1 in neuronal cytoskeleton organization.

Amino Acid Sequence↗

PubMed2002

Ocular development-associated gene (ODAG), a novel gene highly expressed in ocular development.

Complementary DNA (cDNA) arrays were used to detect highly expressed messenger RNA (mRNA) at postnatal day 2 (P2) and P10 in the mouse eye, and several clones highly expressed at P2 were isolated. We focused among them on a novel gene, the ocular development-associated gene (ODAG), which was down regulated at P10. The expression around birth was subsequently confirmed by reverse transcription-polymerase chain reaction. Mouse ODAG cDNA encodes a protein of 266 amino acids. Human ODAG cDNA and genomic structure were identified by basic local alignment search tool analysis of the GenBank database with mouse ODAG. Mouse ODAG-specific mRNA expression was detected in various mouse tissues within the eye at P2 and P7, whereas it was not detected anywhere at P14, suggesting that ODAG may play a role in eye development.

Amino Acid Sequence↗

PubMed2003

Collection, mapping, and annotation of over 28,000 cDNA clones from japonica rice.

We collected and completely sequenced 28,469 full-length complementary DNA clones from Oryza sativa L. ssp. japonica cv. Nipponbare. Through homology searches of publicly available sequence data, we assigned tentative protein functions to 21,596 clones (75.86%). Mapping of the cDNA clones to genomic DNA revealed that there are 19,000 to 20,500 transcription units in the rice genome. Protein informatics analysis against the InterPro database revealed the existence of proteins presented in rice but not in Arabidopsis. Sixty-four percent of our cDNAs are homologous to Arabidopsis proteins.

Alternative Splicing↗

PubMed1998

Identification of novel chicken estrogen receptor-alpha messenger ribonucleic acid isoforms generated by alternative splicing and promoter usage.

Using the rapid amplification of complementary DNA ends (RACE) methodology we have identified three new chicken estrogen receptor-alpha (cER alpha) messenger RNA (mRNA) variants in addition to the previously described form (isoform A). Whereas one of the new variants (isoform B) presents a 5'-extremity contiguous to the 5'-end of isoform A, the two other forms (isoforms C and D) are generated by alternative splicing of upstream exons (C and D) to a common site situated 70 nucleotides upstream of the translation start site in the previously assigned exon 1 (A). The 3'-end of exon 1C has been located at position -1334 upstream of the transcription start site of the A isoform (+1). Whereas the genomic location of exon 1D is unknown, 700 bp 5' to this exon were isolated by genomic walking, and their sequence was determined. The transcription start sites of the cER alpha mRNA isoforms were defined. In transfection experiments, the regions immediately upstream of the A-D cER alpha mRNA isoforms were shown to possess cell-specific promoter activities. Three of these promoters were down-regulated in the presence of estradiol and ER alpha protein. It is concluded, therefore, that the expression of the four different cER alpha mRNA isoforms is under the control of four different promoters. Finally, RT-PCR, S1 nuclease mapping, and primer extension analysis of these different cER alpha mRNA isoforms revealed a differential pattern of expression of the cER alpha gene in chicken tissues. Together, the results suggest that alternative 5'-splicing and promoter usage may be mechanisms used to modulate the levels of expression of the chicken ER alpha gene in a tissue-specific and/or developmental stage-specific manner.

Alternative Splicing↗

PubMed1996

Insulin-like growth factor IEa2 is the predominantly expressed form of IGF in common carp (Cyprinus carpio).

An insulin-like growth factor (IGF) complementary DNA (cDNA) was isolated from a liver cDNA library of adult common carp, Cyprinus carpio. The identity of this cDNA clone was confirmed by nucleotide sequence determination. Sequence comparison with other vertebrate and rainbow trout IGFs showed that this cDNA encodes a particular subtype of IGF-I, IGF-IEa2. A reverse transcription-polymerase chain reaction (RT-PCR) assay showed that this IGF-I is expressed mainly in the liver (hepatopancreas) of adult common carp. Analyses of 26 other cDNA clones isolated from the same library (using a probe carrying the conserved region of the common carp IGF-IEa2 cDNA clone) showed that these clones represent different insert sizes of the same IGF-IEa2. In conclusion, IGF-IEa2 is the predominantly expressed IGF in the liver of adult common carp.

Amino Acid Sequence↗

PubMed1994

Genes encoding acidic and basic class III beta-1,3-glucanases are expressed in tomato plants upon viroid infection.

beta-1,3-glucanases are hydrolytic enzymes considered to constitute part of the general array of defense genes induced by pathogen infection in higher plants. We have isolated and characterized two complementary DNA clones, corresponding to new beta-1,3-glucanases from tomato plants (Lycopersicon esculentum) which are expressed upon challenge with citrus exocortis viroid. Amino acid sequence comparison revealed that they are most similar to beta-1,3-glucanases from tobacco, particularly to PR-Q', the unique component of the class III beta-1,3-glucanase. The deduced amino acid sequences of the two tomato beta-1,3-glucanases indicate that, although being highly similar in amino acid sequence, they have different isoelectric points: pI 10.5 for the basic isoform (Tom PR-Q'b) and pI 5.2 for the acidic one (Tom PR-Q'a). The expression of these two beta-1,3-glucanase messenger RNAs (mRNAs) in response to viroid infection and ethephon treatments was examined. mRNAs for these two isoforms are coordinately expressed and induced similarly to mRNAs for other PR proteins, indicating that they are part of a general and coordinate mechanism of response of tomato plants susceptible to viroid infection.

Amino Acid Sequence↗

PubMed2004

Molecular cloning and functional characterization of fatty acyl desaturase and elongase cDNAs involved in the production of eicosapentaenoic and docosahexaenoic acids from alpha-linolenic acid in Atlantic salmon (Salmo salar).

Fish are the only major dietary source for humans of omega-3 highly unsaturated fatty acids (HUFAs) and with declining fisheries farmed fish such as Atlantic salmon (Salmo salar) constitute an increasing proportion of the fish in the human diet. However, the current high use of fish oils, derived from wild capture marine fisheries, in aquaculture feeds is not sustainable in the longer term and will constrain continuing growth of aquaculture activities. Greater understanding of how fish metabolize and biosynthesize HUFA may lead to more sustainable aquaculture diets. The study described here contributes to an effort to determine the molecular genetics of the HUFA biosynthetic pathway in salmon, with the overall aim being to determine mechanisms for optimizing the use of vegetable oils in Atlantic salmon culture. In this paper we describe the cloning and functional characterization of 2 genes from salmon involved in the biosynthesis of HUFA. A salmon desaturase complementary DNA, SalDes, was isolated that include an open reading frame of 1362 bp specifying a protein of 454 amino acids. The protein sequence includes all the characteristics of microsomal fatty acid desaturases, including 3 histidine boxes, 2 transmembrane regions, and an N-terminal cytochrome b(5) domain containing a heme-binding motif similar to that of other fatty acid desaturases. Functional expression in the yeast Saccharomyces cerevisiae showed SalDes is predominantly an omega-3 delta5 desaturase, a key enzyme in the synthesis of eicosapentaenoic acid (20:5n-3) from alpha-linolenic acid (18:3n-3). The desaturase showed only low levels of delta6 activity toward C(18) polyunsaturated fatty acids. In addition, a fatty acid elongase cDNA, SalElo, was isolated that included an open reading frame of 888 bp, specifying a protein of 295 amino acids. The protein sequence of SalElo included characteristics of microsomal fatty acid elongases, including a histidine box and a transmembrane region. Upon expression in yeast SalElo showed broad substrate specificity for polyunsaturated fatty acids with a range of chain lengths, with the rank order being C(18) > C(20) > C(22). Thus this one polypeptide product displays all fatty acid elongase activities required for the biosynthesis of docosahexaenoic acid (22:6n-3) from 18:3n-3.

Acetyltransferases↗

PubMedDate not supplied

Isolation and identification of 3-methylcrotonyl coenzyme A carboxylase cDNAs and pyruvate carboxylase, and their expression in red seabream (Pagrus major) organs.

We determined complementary DNA sequences of biotin-containing (MCCC1) and non-biotin-containing (MCCC2) subunits of 3-methylcrotonyl coenzyme A carboxylase (MCCase) and pyruvate carboxylase (PCase) using reverse transcriptase polymerase chain reaction of RNA extracted from seabream skeletal muscle and liver. We determined the complete coding sequences of MCCC1 and PC and a partial coding sequence of the major part of MCCC2. Molecular sizes of MCCC1, MCCC2, and PC were 4300, 2400, and 6500 nucleotides, respectively, according to Northern blot analysis. The length of MCCC1 from cDNA sequencing was 4249 nucleotides, indicating the full-length messenger RNA sequence was obtained. Northern blot analyses showed that PC was expressed in muscle, heart, liver, and ovary, but not in spleen. MCCC1 and MCCC2 were expressed at high levels in muscle and ovary, but only trace levels in heart, spleen, and liver. MCCase appears to be particularly important in muscle and ovary, which are active in protein metabolism, while PCase is important in organs active in glycolysis, such as liver.

Amino Acid Sequence↗

PubMed2004

Crassostrea gigas ferritin: cDNA sequence analysis for two heavy chain type subunits and protein purification.

Ferritin has been shown as being the principal iron storage in the majority of living organisms. In marine species, ferritin is also involved in high-level accumulation of (210)Po. As part of our work on the investigation of these radionuclides' concentration in natural environment, ferritin was searched at the gene and protein level. Ferritin was purified from the visceral mass of the oyster Crassostrea gigas by ion-exchange chromatography and HPLC. SDS-PAGE revealed one band of 20 kDa. An Expressed Sequence Tag (EST) library was screened and led to the identification of two complementary DNA (cDNA) involved in ferritin subunit expression. The complete coding sequences and the untranslated regions (UTRs) of the two genes were obtained and a 5' Rapid Amplification of cDNA Ends (RACE) was used to obtain the two iron-responsive elements (IREs) with the predicted stem-loop structures usually present in the 5'-UTR of ferritin mRNA. Sequence alignment in amino acid of the two new cDNA showed an identity with Pinctada fucata (85.4-88.3%), Lymnaea stagnalis (79.3-82.2%) and Helix pomatia (79.1-79.1%). The residues responsible for the ferroxidase center, conserved in all vertebrate H-ferritins, are present in the two oyster ferritin subunits. Oyster ferritins do not present the special characteristics of other invertebrate ferritins like insect ferritins but have some functional similarities with the vertebrate H chains ferritin.

Amino Acid Sequence↗

PubMed2005

Partial cloning of neuronal nitric oxide synthase (nNOS) cDNA and regional distribution of nNOS mRNA in the central nervous system of the Nile tilapia Oreochromis niloticus.

A constitutive NOS complementary DNA (cDNA) was partially cloned by RT-PCR from the brain of a teleost, the Nile tilapia (Oreochromis niloticus), using degenerate primers against conserved regions of NOS. The predicted 206-long amino acid sequence showed a high degree of identity with other vertebrate neuronal NOS (nNOS) protein sequences. In addition, phylogenetic analysis revealed that Nile tilapia NOS clustered with other known nNOS. Using the coupled reaction of semi-quantitative RT-PCR and Southern blotting, the basal tissue expression pattern of the cloned nNOS gene was investigated in discrete areas of the central nervous system (CNS) and in the heart and skeletal muscle tissue. As revealed, expression of nNOS transcripts was detected in all the CNS regions examined, whereas nNOS gene was not expressed in the heart and skeletal muscle. The distribution pattern of nNOS gene expression showed the highest expression levels in the forebrain followed by the optic tectum, the brainstem and the spinal cord, whereas scarce expression was detected in the cerebellum. Cellular expression of nNOS mRNA was analyzed in the CNS by means of in situ hybridization. According to the RT-PCR results, most nNOS mRNA expressing neurons are localized in the telencephalon and diencephalon, whereas in the mesencephalic optic tectum, the brainstem and the spinal cord, nNOS mRNA expressing neurons are relatively more scattered. A very low hybridization signal was detected in the cerebellar cortex. These results suggest that NO is involved in numerous brain functions in teleosts.

Amino Acid Sequence↗

PubMed2002

Isolation and characterization of the murine prostate short-chain dehydrogenase/reductase 1 (Psdr1) gene, a new member of the short-chain steroid dehydrogenase/reductase family.

We report the isolation and characterization of a complementary DNA (cDNA) encoding a novel member of the short-chain dehydrogenase/reductase (SDR) gene family that we have designated murine prostate short-chain dehydrogenase/reductase 1 (Psdr1). Psdr1 was cloned as a 3.2 kbp transcript from mouse testis cDNA based on the sequence of the recently described androgen-regulated human PSDR1 gene (Cancer Res. 61 (2001) 1611). The putative protein encoded by Psdr1 consists of 316 amino acids with 85% identity to human PSDR1. A search against the BLOCKS database of conserved protein motifs indicates that Psdr1 retains features essential for SDR function. Northern analyses demonstrate that Psdr1 is highly expressed in the murine testis and liver and exhibits several isoforms. Cloning and sequence analysis of the putative Psdr1 promoter region identified motifs with homology to the consensus androgen response element and progesterone response element. The Psdr1 gene was mapped to mouse chromosome 12q31-34, which has synteny with the human PSDR1 chromosomal location (14q23-24.3). Together, these data describe a new member of the SDR gene family that may be involved in the tissue-specific metabolism of retinoids or steroid hormones.

Amino Acid Sequence↗